Popularly known as the powerhouse of the cell, mitochondria are not only the site of metabolism and energy generation but also a hub for other cellular processes, including the initiation of cell death. Mitochondrial uptake of the signaling molecule calcium plays a role in the stimulation of ATP production, but too much calcium can lead to opening of the mitochondrial permeability transition pore (mPTP), triggering necrosis. The recent identification of the molecule forming the pore through which calcium can rapidly enter the mitochondria, the mitochondrial calcium uniporter (MCU), has provided a genetic means to directly test the functional importance of calcium uptake. In particular, MCU is part of a large multi-protein complex including other protein components. EMRE and MICU1 are two of these proteins that in cell lines have been shown to play critical roles in regulation of calcium uptake. EMRE was found to be necessary for MCU activity, and its deletion blocked calcium from entering mitochondria. Though its mechanism is controversial, MICU1 has been characterized as a gatekeeper of MCU, inhibiting MCU activity at low levels of extramitochondrial calcium and stimulating MCU when calcium levels rise. This project began with the generation of the first animal models of EMRE and MICU1 deletion. The aims of this project are to determine the impact of EMRE and MICU1 deletion on isolated mitochondria, on primary cells, and on the physiology of the whole animal. The new EMRE and MICU1 knockout mice also make it possible to determine the topology of the calcium uniporter complex, measure effects on basal bioenergetics, and elucidate the role of EMRE and MICU1 respectively in the regulation of mitochondrial calcium uptake and homeostasis. Furthermore, mouse models will reveal the consequences of mitochondrial calcium deregulation on cell death responses, physiological phenotypes including body weight and composition, and disease pathophysiology. Interestingly, human patients with loss-of-function mutations in MICU1 present with conditions such as proximal myopathy and extrapyramidal motor disorder. MICU1 knockout mice are preliminarily observed to exhibit ataxia and tremors, suggesting that this model may mirror human disease features and thus potentially motivate innovative therapies to treat mitochondrial disorder. Altogether, EMRE and MICU1 knockout mice are valuable resources for answering biological questions with both basic and clinical relevance.